Enzyme Kinetics and Metabolic Control Analysis
Example 6.2 Determination of NADH in cell extract using a cyclic enzyme assay
One of the most important applications of enzymes in studies of cell physiology is as a tool to determine
the intracellular concentration of metabolites. As has often been pointed out in Chapters 3 and 5 the
intracellular concentrations of NADH and NAD" and in particular of the ratio NADH/NAD' is of crucial
importance for the cellular behavior.
In lactic bacteria the intracellular concentration of NADH is miniscule - of the order of 1 Mmole (g
- and a very large quantity of cell extract must be used to obtain a reasonable accuracy in
the assay. This is where cyclic assays can come in very handy. For the case of NADH determination
Fig. 6.6 shows that the redox reaction between NADH and NAD" is coupled to two other redox reactions,
the oxidation of ethanol to acetaldehyde catalyzed by the enzyme alcohol dehydrogenase (ADH), and
the reduction by NADH of an indicator, MTT for which the reduced form absorbs light at 570 nm. As
long as there is ethanol and MTT0X
present the absorbance signal will continue to increase. Since both
substrates are available in surplus during the whole experiment, and since NADH is continuously being
regenerated by the oxidation of ethanol its concentration stays constant. As the reaction of NADH with
MTT,„ is first order in the NADH concentration the slope of the absorbance versus time curve is
proportional to the amount of NADH initially present in the sample.
Figure 6.6 A cyclic assay in which the net reaction is: Ethanol + MTT0X
Acetaldehyde + MTT [ed
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